PROJECT SUMMARY Myelodysplastic syndromes (MDS) are disorders of hematopoietic stem and progenitor cells (HSPCs) that results in cytopenias, however, the genetic causes of this dysfunctional hematopoiesis remain unclear. Almost all patients suffer anemia, a major cause of morbidity, mortality, and health care costs. Deletion of part or all of chromosome 7 [-7/del(7q)] is a common cytogenetic abnormality in MDS and carries a poor prognosis. We identified CUX1, a homeodomain-containing transcription factor encoded on 7q, to be frequently inactivated in myeloid diseases. We reported that CUX1 has highly conserved regulatory functions in both human and Drosophila blood cells. CUX1 inactivating mutations have since been reported in MDS and are independently associated with a poor prognosis. We have now generated an innovative Cux1 knockdown mouse model. Cux1-knockdown mice develop a spontaneous myeloproliferative disorder with many features of human MDS, including megakaryocyte, granulocyte, and erythroid dysplasia, and a fatal anemia. Preliminary studies indicate that Cux1 knockdown impacts multiple stages of hematopoiesis, disrupting early HSPC functions as well as blocking late stages of erythroid development. A major knowledge gap, which this proposal is designed to address, is the mechanism by which CUX1 regulates normal hematopoiesis. The overall objective is to determine the transcriptional role for CUX1 in normal HSPCs and erythroid progenitors and the pathways downstream of CUX1 haploinsufficiency that block erythroid differentiation. Aim 1: Hypothesis ? CUX1 is a transcriptional regulator of HSPC homeostasis conserved in mice and humans. We will take advantage of our novel mouse model for in vivo analyses of Cux1 regulation of HSPC quiescence, proliferation, and differentiation. We will perform complementary studies with primary human HSPCs to establish the translational relevance of this work. We will capitalize on leading-edge functional genomics approaches to identify CUX1 genomic targets in human HSPCs. Aim 2: Hypothesis ? CUX1 promotes erythroblast cell cycle exit necessary for terminal differentiation by repressing PI3K signaling. We will identify the specific developmental stage of erythropoiesis disrupted by Cux1 knockdown in mice. We will determine the conserved role for CUX1 in human red cell development, and the pathways induced by CUX1 deficiency that disrupt erythropoiesis. Our functional and genomic analyses of primary human HSCs will dovetail with in vivo assays to elucidate the critical role for CUX1 transcriptional regulation of normal erythropoiesis. This work will have a positive impact on the fields of HSC and erythroid biology by identifying the molecular mechanism by which CUX1 regulates normal HSC functions and the pathogenesis of CUX1-deficiency in disease states. Our studies will reveal novel therapeutic targets for treating anemia in MDS patients and a murine model of MDS for preclinical studies.